Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

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of the initiator methionyl-tRNA

Marie Naveau, Christine Lazennec-Schurdevin, Michel Panvert, Etienne Dubiez, Yves Mechulam, Emmanuelle Schmitt

To cite this version:

Marie Naveau, Christine Lazennec-Schurdevin, Michel Panvert, Etienne Dubiez, Yves Mechulam, et

al.. Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA. Nucleic

Acids Research, Oxford University Press, 2013, 41 (1), pp.1047-1057. �10.1093/nar/gks1180�. �hal-

00840386�

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Roles of yeast eIF2a and eIF2b subunits in the binding of the initiator methionyl-tRNA

Marie Naveau, Christine Lazennec-Schurdevin, Michel Panvert, Etienne Dubiez, Yves Mechulam and Emmanuelle Schmitt*

Laboratoire de Biochimie, Unite´ mixte de Recherche 7654, Ecole Polytechnique, Centre National de la Recherche Scientifique, F-91128 Palaiseau Cedex, France

Received July 17, 2012; Revised October 19, 2012; Accepted October 26, 2012

ABSTRACT

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was exten- sively studied in the archaeal system. Thecsubunit is able to bind tRNA, but theasubunit is required to reach high affinity whereas thebsubunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with b having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2c subunit, we designed chimeric eIF2 by assembling yeast a and b subunits to archaeal c subunit. We show that theb subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The asubunit apparently has a modest contribution. However, the positive effect ofaon tRNA binding can be progressively increased upon shortening the acidic C-terminal extension.

These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the a subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex.

INTRODUCTION

In eukaryotic and archaeal cells, the initiator tRNA carrier is the eukaryotic/archaeal translation initiation factor 2 (e/aIF2) heterotrimer. In its GTP-bound form, this factor speciﬁcally binds Met-tRNAiMet

and handles it in the translation initiation complex. After start codon recognition, the factor, in its GDP-bound form, loses afﬁnity for Met-tRNAiMet

and eventually dissociates from the initiation complex. This leaves Met-tRNAiMet

in the P-site of the small ribosomal subunit and allows the ﬁnal steps of initiation to occur (1,2). In this process, speciﬁc binding of the initiator tRNA by e/aIF2 is crucial for accuracy. In both archaea and eukaryotes, the Kd

values of the e/aIF2–GDPNP–Met-tRNAiMet

complexes are in the nanomolar range (3–8).

e/aIF2 results from the association of three subunits,a, b and g. In archaea, the heterotrimer consists of a rigid central part, formed by the g subunit, the C-terminal domain 3 of the a subunit and the N-terminal helix of the b subunit. Two mobile parts formed by domains 1 and 2 of the a subunit, and by the a–b and the zinc- binding domains of the b subunit are appended to the central core (Figure 1A) (6,9–12).

Until very recently, based on high structural resem- blance of the g subunit with the elongation factor EF1A as well as on site-directed mutagenesis studies, it was believed that the binding mode of the tRNA molecule on aIF2 was similar to that observed with the elongation factor (3,7,9,13,14). However, biochemical results and de- termination of the 5-A˚ crystal structure of archaeal Sulfolubus solfataricus IF2 (Ss-aIF2) bound to initiator methionyl-tRNA have broken this model (15,16). In the 3D structure of the aIF2–GDPNP–tRNA complex, the tRNA is bound by the a and g subunits of aIF2 (Figure 1A). aIF2 approaches tRNA from the acceptor stem minor groove side, whereas EF1A approaches tRNA from the T-stem minor groove side. Despite this, thanks to a kinked conformation, the acceptor end of the tRNA fits in a channel on aIF2g, which corresponds to the tRNA acceptor end-binding channel on EF1A. This model clearly explains why the isolated g subunit of archaeal aIF2 is indeed able to bind initiator methionyl- tRNA but with a binding affinity highly reduced when compared with the tRNA-binding affinity for the complete aIF2 heterotrimer. Indeed, the a subunit provides the heterotrimer with almost its full tRNA- binding affinity, whereas the b subunit only slightly contributes to tRNA binding (5–7,15).

In contrast with archaeal aIF2, the construction of a Saccharomyces cerevisiae strain completely lacking eIF2a

*To whom correspondence should be addressed. Tel: +33 1 69334885; Fax: +33 1 69334909; Email: emma@botrytis.polytechnique.fr ßThe Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

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and allowing puriﬁcation of an eIF2bg heterodimer has shown that, in yeast,a would only slightly contribute to tRNA-binding afﬁnity (no more than a factor of 5) (17), whereas b would have an important effect (18). From these data, an ‘eukaryotic behavior’, with a major role for the b subunit in the binding of the tRNA and a minor role for the a subunit would be opposed to an

‘archaeal behavior’ in whichahas the major contribution.

Hence, the possibility that the structural involvement of the peripheral subunits in the eukaryotic ternary initiation complex differs from that in the archaeal one cannot be completely excluded. Notably, eIF2 from the primitive eu- karyoteEncephalitozoon cuniculi displays an intermediate behavior with a and b subunits equally contributing to tRNA-binding afﬁnity (19).

Functional dissection of S. cerevisiae eIF2in vitrowas impaired by the inability to produce the isolatedgsubunit.

However, very recently, we reported that a chimeric protein formed by assembling the g subunit of S. solfataricus aIF2 with the a subunit of S. cerevisiae eIF2 was useful to study the role of the yeast eIF2a subunit (15). Therefore, chimeric proteins appear as an attractive tool for studying the role of the yeast peripheral subunits of eIF2 in tRNA binding. In the present study, using chimeric e/aIF2, we show that thebsubunit of yeast has indeed an important role in tRNA-binding afﬁnity.

The N- and C-terminal domains of yeast eIF2bare necessary to obtain full tRNA-binding afﬁnity, the C-terminal extension ofbhaving the most important role. The effect of the bsubunit on tRNA-binding afﬁnity may be either C

N

C

βh1 γDI γDII

αD3

αD1 αD2

1 93 178

β-barrell α−β domain

266

1 85 175

4 0 3 5 7 2 1

5 r e S

1 125 234

139

1/4 104 e/aIF2β

272/285 17

α1 ZBD

142

S. solfataricus S. cerevisiae

e/aIF2α S. solfataricus

S. cerevisiae B

A

19

33

γDIII

139

helical domain

α−β domain

Figure 1. Eukaryotic and archaeal e/aIF2. (A) Cartoon representation of Ss-aIF2 in complex with GDPNP and Met-tRNAfMet. The cartoon was drawn from PDB ID 3V11 (15). The color code is as follows: G-domain ofg(gDI, 1–210) in green, domain II (gDII, 211–327) in yellow, domain III (gDIII, 328–415) in orange, domain 1 ofain dark blue (aD1, 1–85), domain 2 ofain blue (aD2, 86–174), domain 3 ofain cyan (aD3, 175–266).

The N-terminalahelix of thebsubunit (3–19) anchored togDI is colored in pink. Note that the position of the rest of thebsubunit (residues 33–139, encircled with a dotted line) is only a tentative model, derived from SAXS data (15). The ﬁgure was drawn with PyMOL (http://www.pymol.

org). (B) Schematic structural organizations of e/aIF2aand bsubunits. The colored boxes indicate the structural domains. Speciﬁc eukaryotic domains and extensions are colored in orange. Gray bars symbolize the K-boxes in the N-terminal domain of yeast eIF2b.

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direct or indirect. The intact a subunit apparently only slightly contributes to tRNA binding. However, shortening of the acidic C-terminal extension revealed a positive effect of thea subunit on tRNA binding (15). Here, we show that the negative effect of the C-terminal extension is directly related to the size of the acidic C-terminal tail.

Moreover, the small-angle X-ray scattering (SAXS) diffusion curve obtained with the Ch-eIF2aCg–GDPNP–

met-tRNAfMet

complex agrees well with the theoretical curve computed from the crystallographic structure of Ss-aIF2ag bound to GDPND and met-tRNAfMet

. Therefore, altogether, the results are compatible with the idea that, in yeast eIF2, the tRNA molecule is bound in an orientation similar to that observed in the archaeal aIF2–

GDPNP–tRNA complex.

MATERIALS AND METHODS

Cloning, expression and production of yeast eIF2a and bsubunits and their variants

The genes encoding the a and b subunits of eIF2 from S. cerevisiaewere ampliﬁed by PCR from genomic DNA and cloned in pET-derivative vectors. The gene coding for thea subunit was cloned between the NdeI and BamHI restriction sites of pET15b. The resulting plasmid called pET15bY-a led to the expression of an N-terminally tagged version of yeastasubunit in Escherichia coli. The gene coding for the b subunit was cloned between the NdeI and SacII restriction sites of pET3a to give pET3aY-b. This plasmid allows expression of an unmodi- ﬁed version of yeast eIF2b.

Deletions in the target genes were carried using the QuikChange Site-directed Mutagenesis method (Stratagen). The full sequence of all mutated genes was conﬁrmed by resequencing. Deletion of the C-terminal extension of yeast eIF2a was achieved using pET15bY-a to give various truncated forms. pET15bY-aC274 produced a protein ending at residue T274 (15).

pET15bY-aC283 produced a protein ending at residue L283, pET15bY-aC292 produced a protein ending at residue S292 and pET15bY-aC298 produced a protein ending at residue E298. The DNAs coding for yeastaD3 or yeast aD3C274 were obtained by the QuikChange Site-directed Mutagenesis method with the introduction of a start codon at position 178.

Deletion of the C-terminal domain of yeast eIF2bwas achieved using pET3aY-bto give pET3aY-bC. The construction allowed expression of a b subunit ending at residue I271. pET15bY-b allowed expression of an N-terminally tagged version of yeast eIF2b. pET15bY- bN was a derivative of pET15bY-b producing a b subunit deleted of its N-terminal domain (residues 1–125) His-tagged at its N-terminus. The doubly deleted mutant eIF2bNC was expressed from pET15bbNC, a derivative of pET15bbY-N. The resulting protein corresponded to an N-terminally histidine-tagged version of yeast eIF2bcomprised of the core domain of eIF2b, from residue E126 to I271.

Each subunit was overexpressed separately in E. coli BL21 Rosetta pLacI-Rare (Merck, Novagen). One-liter

cultures were in 2xTY containing 50mg/ml of ampicillin and 34mg/ml of chloramphenicol. Expression was induced after an overnight culture at 37C (OD6502.5) by adding 1 mM of IPTG. After induction, the cultures were continued for 5–6 h at 18C.

Purification of chimeric eIF2

Overexpression of Ss-aIF2g subunit was performed as described (6). Cultures of cells each overproducing one of the three subunits (250 ml for yeast a, 500 ml for yeast b and 500 ml for S. solfataricus aIF2g) were harvested, mixed in 80 ml of buffer A (10 mM HEPES pH 7.5, 500 mM NaCl, 3 mM 2-mercaptoethanol, 0.1 mM PMSF and 0.1 mM benzamidine) and disrupted by sonication. After centrifugation, the supernatant was loaded onto a column (3 ml) containing Talon affinity resin (Clontech) equilibrated in the same buffer. The resin was first washed with 50 ml of buffer A and then with 50 ml of buffer A supplemented with 10 mM imidazole. The fractions containing the heterotrimer were finally recovered after elution with buffer A containing 125 mM imidazole.

To remove the excess of the yeasta subunit, the afﬁnity column eluate was diluted to 300 mM NaCl and then loaded onto a 5 ml S-Hiload column (10 mm5 cm; GE Healthcare) equilibrated in buffer B (10 mM HEPES pH 7.5, 300 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM PMSF and 0.1 mM benzamidine). A gradient from 300 mM NaCl to 600 mM NaCl was used for elution (128 ml at a ﬂow rate of 2 ml/min). After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE) analysis, the fractions containing the heterotrimer were pooled and dialyzed against buffer B. Finally, the protein was concentrated by using Centricon 30 concentrators. Either GDPNP-Mg²⁺ (1 mM) or GDP-Mg²⁺

(1 mM) was added before storage at 4C of the recovered heterotrimer (Figure 2, lane 1). The same procedure was applied to purify the Ch-eIF2aCbg, Ch-eIF2bNg and Ch-eIF2bNCg (Figure 2, lanes 4, 6 and 5).

Purification of Ch-eIF2bcand Ch-eIF2b"Cc

Cultures of cells each overproducing one of the two subunits (500 ml for yeastbor yeast bC and 500 ml for S. solfataricus aIF2g) were harvested, mixed in 40 ml of buffer B (10 mM HEPES pH 7.5, 300 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM PMSF and 0.1 mM benzamidine) and disrupted by sonication. After centrifugation, the supernatant was loaded onto a 15 ml S-Hiload column (16 mm20 cm; GE Healthcare) equilibrated in buffer B. A gradient from 300 mM NaCl to 600 mM NaCl was used for elution (120 ml at a ﬂow rate of 2 ml/

min). After SDS–PAGE analysis, the fractions containing the heterodimer were pooled and dialyzed against buffer B. To remove remaining nucleic acids, the recovered protein was then loaded onto a 15 ml Q-Hiload column (16 mm20 cm; GE Healthcare) equilibrated in buffer B.

The heterodimer was recovered in the ﬂow-through fraction and then concentrated by using Centricon 30 concentrators. Either GDPNP-Mg²⁺(1 mM) or GDP-Mg²⁺

(1 mM) was added before storage at 4C of the recovered heterodimer (see Figure 2, lane 3 for puriﬁed Ch-eIF2bg).

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Purification of Ch-eIF2acheterodimers and of its variants The procedure used for the puriﬁcation of Ch-eIF2ag heterodimer was described previously (15) (Figure 2, lane 2). To purify the Ch-eIF2aCg heterodimer variants (aC274, aC283, aC292 and aC298), the S-Hiload step was replaced by a molecular sieve using a Superdex 200 HR 10/30 column (GE Healthcare). The puriﬁed proteins (Figure 2, lane 7) were stored in buffer B in the presence of either 1 mM GDPNP-Mg²⁺or 1 mM GDP-Mg²⁺.

The procedure used to purify Ch-eIF2aD3g and Ch-eIF2aD3Cg heterodimers was the same as that used for Ch-eIF2aCg heterodimer variants, except that after the Talon column, size-exclusion chromatography on Superdex 75 HR 10/30 (GE Healthcare) was used to polish the preparation.

Ch-eIF2aCg and Ch-eIF2aD3Cg protein used to perform SAXS studies were purified as follows. aC andaD3C were first purified using Talon affinity resin.

The His-tag extensions of the recovered proteins were then removed during overnight dialysis in a buffer containing 10 mM HEPES pH 7.5, 200 mM NaCl, 3 mM 2-mercaptoethanol, 10 mM CaCl2 and Thrombin (0.25 U/mg of substrate protein) at 4C. The g subunit of S. solfataricus was purified as described (6). aC and/or aD3C was assembled with Ss-aIF2g and the heterodimers were finally purified using a last step of molecular sieving using a Superdex 200 HR 10/30 column (GE Healthcare) in buffer C (10 mM HEPES pH 7.5, 200 mM NaCl and 3 mM 2-mercaptoethanol).

Purification of Ch-Encc-eIF2acand of Ch-Encc-eIF2a"Cc

BL21 Rosetta pLacI-Rare containing the pET28b+gtc- Encc plasmid was used to overexpress the g subunit from E. cuniculi (19). The resulting protein carried a 6-histidine tag at its C-terminus. One-liter cultures were in 2xTY containing 25mg/ml of kanamycin and 34mg/ml of chloramphenicol. Expression was induced by adding 1 mM of IPTG when OD650reached 0.8. After induction, the cultures were continued for 8–12 h at 18C.

Cultures of cells each overproducing one of the two subunits (250 ml for yeast a, 500 ml for E. cuniculi eIF2g) were harvested, mixed in 40 ml of buffer A and

disrupted by sonication. After centrifugation, the supernatant was loaded onto a column (3 ml) containing Talon afﬁnity resin (Clontech) equilibrated in the same buffer.

The resin was first washed with 50 ml of buffer A and then with 50 ml of buffer A supplemented with 10 mM imidazole. The fractions containing the heterodimer were finally recovered after elution with buffer A containing 125 mM imidazole. To remove the excess of the yeast a subunit, the affinity column eluate was diluted to 250 mM NaCl and then loaded onto a 5 ml Q-Sepharose HP column (10 mm5 cm; GE Healthcare) equilibrated in buffer C (10 mM HEPES pH 7.5, 250 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM PMSF and 0.1 mM benzamidine). The heterodimer flowed through the column whereas the excess yeast a subunit was retained.

After SDS–PAGE analysis, the fractions containing the heterodimer were pooled and dialyzed against buffer B.

Finally, the protein was concentrated by using Centricon 30 concentrators. Either GDPNP-Mg²⁺ (1 mM) or GDP-Mg²⁺(1 mM) was added before storage at 4C of the recovered heterodimers.

The same procedure was used to purify ChEncc- eIF2aCg except that after the Talon column, the protein was loaded onto a Superdex 75 column (HR10/

30, GE Healthcare) equilibrated in buffer B. The eluted heterodimer was loaded onto a 5 ml S-Sepharose HP column (10 mm5 cm, GE Healthcare) equilibrated in buffer D (10 mM HEPES pH 7.5, 200 mM NaCl, 10 mM 2-mercaptoethanol, 0.1 mM PMSF and 0.1 mM benzamidine). A gradient from 200 to 800 mM NaCl was used for elution (80 ml at a ﬂow rate of 2 ml/min).

The fractions containing the heterodimer were pooled and dialyzed against buffer B. Finally, the protein was concentrated by using Centricon 30 concentrators.

Either GDPNP-Mg²⁺ (1 mM) or GDP-Mg²⁺ (1 mM) was added before storage at 4C of the recovered heterodimer (Figure 2, lanes 8 and 9).

Protection assay

The protocol used was derived from (20). tRNAfMet

was produced in E. coli from synthetic genes and puriﬁed as described (21,22). Endogenous tRNAiMet

puriﬁed from S. cerevisiae was a generous gift of Dr Ge´rard Keith (Institut de Biologie Mole´culaire et Cellulaire,

Y-α Y-β Ss-γ

Y- α∆C274

Encc-γ

Y-α∆C274 Y-α

8 9

Y-β∆N Y-β∆N∆C γ^Ss Y-α∆C274

5 6 7

1 2 3 4

Figure 2. SDS–PAGE analysis of puriﬁed Ch-eIF2 variants. The 12.5% SDS–PAGE were stained with Coomassie Blue. Lane 1, Ch-eIF2 heterotrimer. Lane 2, Ch-eIF2ag heterodimer. Lane 3, Ch-eIF2bgheterodimer. Lane 4, Ch-eIF2(aC274)bg. Positions of tagged yeast eIF2a (Y-a, 369 kDa), tagged yeast eIF2aC274 (Y-aC, 334 kDa), yeast eIF2b(Y-b, 314 kDa) and Ss-aIF2g(Ss-g, 456 kDa) are indicated. Lane 5, Ch-eIF2bNCg heterodimer. Lane 6, Ch-eIF2bNg heterodimer. Lane 7, Ch-eIF2aC274g heterodimer. Positions of yeast eIF2bNC (Y-bNC), eIF2bN (Y-bN) and eIF2aC274 (Y-aC) are indicated. Lane 8, ChEncc-eIF2(aC274)gheterodimer, Lane 9, ChEncc-eIF2ag heterodimer. Positions of tagged yeast eIF2aC274 (Y-aC, 334 kDa), tagged yeast eIF2a (Y-a, 369 kDa) and E. cuniculi eIF2g (Encc g, 489 kDa) are indicated.

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Strasbourg, France). Full aminoacylation with [³⁵S]-methionine (10 000 dpm/pmol; Perkin Elmer) was achieved using homogeneous E. coli M547 MetRS (23).

tRNAfMet

UAC, a derivative of tRNAfMet

carrying a UAC (Val) anticodon was produced as described (21,22).

Valylation with [¹⁴C]Val (563 dpm/pmol) was performed with homogeneous E. coli valyl-tRNA synthetase as described (7). Aminoacyl-tRNAs were precipitated with ethanol in the presence of 0.3 M NaAc pH 5.5 and stored at 20C in 100% EtOH in small aliquots.

Before use, aminoacylated tRNAs were redissolved in water and full aminoacylation was systematically controlled through measurement of radioactivity after precipitation in 5% trichloroacetic acid (TCA).

Protection by chimeric eIF2 (Ch-eIF2) variants of methionyl-tRNAfMet

against spontaneous hydrolysis was assayed as follows. Reaction mixtures (150ml) contained 20 mM HEPES–NaOH pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA (bovine serum albumin; ROCHE), 5% glycerol, 0.1%

triton X-100, 1 mM GDPNP and 2 nM of E. coli methionyl-tRNAfMet

. The concentrations of the proteins were determined from A280 measurements using extinc- tion coefﬁcients computed from the amino acid sequences.

Concentrations of Ch-eIF2 variants were varied from 1 nM to 30mM, using a range depending on theKd value to be measured. The mixtures were incubated at 30C. To determine the rate constants of deacylation, 20ml aliquots were withdrawn at various times (usually, six aliquots from 5 to 60 min) and precipitated in 5% TCA in the presence of 80mg of yeast RNA as carrier. In all cases, the deacylation curve as a function of time could be fitted with a single exponential. For eachKdmeasurement, a set of 8–10 experiments corresponding to 8–10 different protein concentrations was performed. The rate constants measured at variable protein concentrations were then fitted to simple binding curves (20) from which the dissociation constant of the studied protein–tRNA complexes and their associated standard errors could be deduced using the MC-Fit program (24). Each experiment was independently repeated at least twice, without significant variation of the results.

Small-angle X-ray scattering

SAXS experiments were conducted on the SWING beamline at the SOLEIL synchrotron as described (15).

For these experiments, the His-tags of eIF2a3C and of eIF2aC were removed before assembly with Ss-aIF2g, as described above. A concentrated sample of Ch-eIF2(a3C)g (4 nmol in 40ml) was injected onto a size-exclusion column (agilent Bio column SEC3; 300 A˚) using an Agilent HPLC system and eluted directly into the SAXS ﬂow-through capillary cell at a ﬂow rate of 0.4 ml/

min.

To collect data on Ch-eIF2(aC)g–GDPNP–

Met-tRNAfMet

complex, the protein was mixed with a 1.15-fold molar excess of Met-tRNAfMet

and injected as a concentrated sample (20 nmol in 250ml) onto a size-exclusion column (Superdex 200 HR10/30, GE Healthcare), using an Agilent HPLC system and eluted

directly into the SAXS ﬂow-through capillary cell at a ﬂow rate of 0.4 ml/min. The same procedure was used to collect data on Ss-aIF2–GDPNP–Met-tRNAfMet

, Ss-aIF2–GDPNP–Met-tRNAfMet

(A1–U72) and Ss-aIF2–

GDPNP–Ss-Met-tRNAiMet

complexes. tRNAfMet

(A1–

U72) and Ss-tRNAiMet

were puriﬁed and aminoacylated as described (7,25).

For all experiments, the elution buffer consisted of 10 mM MOPS (pH 6.7), 200 mM NaCl and 5 mM MgCl2. SAXS data were collected continuously, with a frame duration of 1.5 s and a dead time between frames of 1.0 s. Selected frames corresponding to the main elution peak were averaged (26). A large number of frames were collected during the ﬁrst minutes of the elution, and these were averaged to account for buffer scattering, which was subsequently subtracted from the signal during elution of the protein or protein–tRNA complex. Data reduction to absolute units, frame averaging and subtraction were done using FOXTROT (26). All subsequent data processing, analysis and modeling steps were carried out with PRIMUS and other programs of the ATSAS suite (27).

Scattered intensity curves were calculated from the atomic coordinates of the crystallographic structure, using CRYSOL with 50 harmonics (28). This program was also used to ﬁt the calculated curve to the experimental one, by adjusting the excluded volume, the averaged atomic radius and the contrast of the hydration layer sur- rounding the particle in solution.

RESULTS

Production and assembly of the Ch-eIF2 heterotrimer The chimeric heterotrimer (Ch-eIF2) was formed by assembling the archaeal g subunit ofS. solfataricus aIF2 with theaandbsubunits ofS. cerevisiaeeIF2. To facili- tate purification of Ch-eIF2, we used an N-terminally tagged version of yeast eIF2a and native versions of yeast eIF2b and Ss-aIF2g. The three subunits were produced independently in E. coli. The first step of purification of the assembled Ch-eIF2 heterotrimer was a metal affinity chromatography. This step showed that the archaeal g subunit was able to interact with both yeast a and b subunits. To remove the excess of the yeastasubunit, we then used anion exchange chromatography. This purification step allowed to recover a correct stoechiometry between the three subunits, as judged by SDS–PAGE analysis (Figure 2, lane 1).

Binding of tRNAiMet

onto e/aIF2 leads to protection of aminoacylated tRNA against spontaneous deacylation.

Thus, dissociation constants of Met-tRNAiMet

from an e/

aIF2–GDPNP–Met-tRNA_i^Met complex can be estimated by following deacylation rates in the presence of various eIF2 concentrations at a ﬁxed tRNA concentration (7,20).

The sequence of theE. coliinitiator tRNAfMet

has a high homology with the one from the archaeal initiator tRNA (Figure 3). In particular, the acceptor stems of both tRNAs are identical, with the exception that the A1–U72 base pair in the archaeal tRNA is replaced by C1–A72 in the bacterial one. Moreover, it was previously shown thatE. coliMet-tRNAfMet

was as good a ligand of

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archaeal aIF2 as the cognate initiator Ss-tRNAiMet

produced inE. coli(15).To further validate at a structural level, the use ofE. coliinitiator Met-tRNAfMet

as a model ligand, we compared the SAXS curve of Ss-aIF2–

GDPNP–Met-tRNAfMet

with the corresponding ones with either Met-tRNAfMet

A1–U72 variant or Met- tRNAiMet

from S. solfataricus. The three SAXS curves were nicely superimposable (Supplementary Figure S1), showing that the three complexes were highly similar.

Escherichia coliMet-tRNAfMet

initiator tRNA was used as a model ligand to study tRNA binding by Ch-eIF2.

Ch-eIF2 bound efficiently initiator methionyl-tRNA in the presence of GDPNP-Mg²⁺ with a Kd value of 87 ± 10 nM (Table 1, row 1). Moreover, in the presence of GDP instead of GDPNP, theKdvalue was increased to 700 ± 300 nM. Therefore, GDP lowered the binding affinity of the initiator methionyl-tRNA by one order of magnitude, in keeping with the nucleotide effects observed with yeast eIF2 [factor of 20 (4)] or Ss-aIF2 [factor of 100, (6)]. Furthermore, we verified that Ch-eIF2 could still interact with authentic yeast Met-tRNAiMet

(Kd= 27 ± 5mM; Table 1, row 1; Figure 3). The binding afﬁnity of both initiator tRNAs for the chimeric factor in the presence of GDPNP were reasonably high, when compared with the afﬁnity for eIF2 from yeast [Kd= 9 nM, (4)] or for Ss-aIF2 [Kd= 1.5 nM, (6)]. The observed discrepancies between the Kd values measured with yeast eIF2, Ss-aIF2 or Ch-eIF2 could be in part due to differences in the assay conditions. Indeed, in the case of yeast eIF2 the temperature used in the assay was 26C and in the case of Ss-aIF2, the temperature used in the assay was 51C. Here, to avoid thermal effects on yeast aandbproteins, we performed the measurements at 30C, a temperature sub-optimal for the archaeal g subunit of Ch-eIF2 (5). Finally, it is known that archaeal and eukaryotic eIF2 strongly recognize the methionine moiety on initiator tRNAs (4,7,29,30). Therefore, as an additional

control to validate the use of Ch-eIF2 as a tool, we veriﬁed that the chimeric heterotrimer had the same property. We used Val-tRNAfMet

UAC in a standard protection assay. Instead of protection against deacylation, we observed a surprising 2-fold increase of the rate of deacylation in the presence of 7.4mM Ch-eIF2. Possibly, low-afﬁnity unspeciﬁc binding of the Val-tRNAfMet

UAC on the factor may favor hydrolysis of the esteriﬁed amino acid. Nevertheless, this experiment allowed to conclude that the presence of the valyl group strongly affected speciﬁc aminoacyl-tRNA binding.

The yeastb subunit strongly influences tRNA binding on Ch-eIF2

At 30C, only weak, though signiﬁcant, GDPNP- dependent binding of initiator tRNA to Ss-aIF2g occurs (Table 1, row 2). Therefore, the large difference between the Kd value measured with the Ss-g subunit alone and that measured for the full Ch-eIF2 trimer showed that thea and/or theb subunits participate in tRNA binding (Table 1, rows 1 and 2). In order to examine the contribution of each peripheral subunit, Ch-eIF2ag and Ch-eIF2bg heterodimers were produced and puriﬁed (Figure 2, lanes 2 and 3). Using Ch-eIF2bg, theKdvalue of Met-tRNAfMet

was 55 ± 10 nM (Table 1, row 3) whereas using Ch-eIF2ag, the Kd value of Met-tRNAfMet

was 9200 ± 2000 nM (Table 1, row 7).

Hence, theb subunit of yeast eIF2 improved the binding afﬁnity of Met-tRNAfMet

by three orders of magnitude (Table 1, rows 2 and 3, Supplementary Figure S2A). The yeastasubunit also participated in the binding afﬁnity of Met-tRNAfMet

. Its contribution was however two orders of magnitude less important than that of the b subunit (Table 1, rows 2 and 7, Supplementary Figure S2A).

Notably, in the absence of the a subunit, the afﬁnity of Ch-eIF2bg for Met-tRNAfMet

was similar to that

Figure 3. Cloverleaf representations of initiator Met-tRNAs fromS. solfataricus,E. coliandS. cerevisiaecytoplasm. Nucleotides denoted with gray boxes are universally conserved in initiator tRNAs. Post-transcriptional modiﬁcations are indicated in the cases ofE. coliandS. cerevisiae.

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measured with the complete chimeric heterotrimer (compare rows 1 and 3, Table 1 and Supplementary Figure S2A). The same effects of the a and b subunits were observed using authentic yeast Met-tRNAiMet

as a substrate (Table 1, rows 2, 3 and 7). This indicated that the association of thebsubunit togwas enough to retrieve almost the same binding afﬁnity for tRNA as full Ch-eIF2. Ch-eIF2bgremained speciﬁc of the methionine moiety of the tRNA ligand. Indeed, the presence of 10mM Ch-eIF2bg only decreased the deacylation rate of Val-tRNAfMet

UAC by 20%, consistent with a dissociation constant of the order of several tens of micromolar.

Notably, the behavior of the yeast peripheral subunits was opposite to that observed with archaeala andbsubunits (Supplementary Figure S2A).

N- and C-terminal extensions of yeast eIF2bare necessary for Met-tRNAfMet

binding

The yeastbsubunit is made of a conserved structural core corresponding to the archaeal version of the protein to which are appended eukaryote-speciﬁc domains, namely, an N-terminal domain and a short C-terminal extension (Figure 1B). To evaluate the roles of the yeast-eIF2bex- tensions in tRNA binding, three truncated versions of the subunit were produced. In eIF2bN, the ﬁrst 125 residues were removed. In eIF2bC, residues 272–285 were removed. The two deletions were also performed simul- taneously giving eIF2bNC.

The three heterodimers Ch-eIF2bNg, Ch-eIF2bCg and Ch-eIF2bNCg were puriﬁed and Met-tRNAfMet

- binding afﬁnities were measured. As shown in Table 1, the N-terminal domain contributed by a factor of 4.2 to tRNA-binding afﬁnity (rows 3 and 4) and the C-terminal extension of eIF2b contributed by a factor of 9.7 (rows 3 and 5). These results showed that both eukaryotic extensions participate in the binding of the tRNA molecule with the contribution of the short basic C-terminal extension being twice as much as that of the

N-terminal domain. Moreover, with the double truncation (Ch-eIF2bNCg), the dissociation constant fell down by a factor of 63 when compared with unmodiﬁed Ch-eIF2bg(rows 3 and 6). Therefore, the positive effects of both extensions on the binding of the initiator tRNA were cumulative (Supplementary Figure S2B). Finally, using Ch-eIF2bNCg, a Kd-value for Met- tRNAfMet

binding of 3500 ± 1400 nM was measured. The afﬁnity of the tRNA remained 15-fold higher than that with Ss-aIF2g alone. Hence, the conserved core of yeast eIF2bstill contains some features allowing it to participate in tRNA-binding afﬁnity (Supplementary Figure S2B).

The acidic C-terminal extension of yeast eIF2a interferes with tRNA binding

The yeastasubunit of eIF2 comprises a conserved structural core corresponding to the archaeal version of the protein, with an additional highly acidic (pI = 3.14) C-terminal tail, containing 16 Asp or Glu out of 30 residues.

This acidic C-terminal extension is characteristic of eukaryotic eIF2a (Figures 1B and 4). Previously, the C-terminal region corresponding to residues 275–304 was removed through site-directed mutagenesis of the yeast eIF2a gene (15). With the heterodimer Ch-eIF2aC274g, a Kd-value for the initiator methionyl-tRNA of 73 ± 10 nM was measured (15).

Therefore, removal of the C-terminal tail increased the binding afﬁnity by more than two orders of magnitude (rows 7 and 8 in Table 1 and Supplementary Figure S2C). The same effect was observed using authentic yeast Met-tRNA_i^Met as a substrate (K_d= 36 ± 8 nM;

Table 1). Hence, the C-terminal extension of the yeast a subunit is strongly unfavorable to the binding of the tRNA on Ch-eIF2ag. Again, we veriﬁed that Ch-eIF2aC274g remained speciﬁc of the methionine moiety of the tRNA ligand. Indeed, the presence of 10mM Ch-eIF2aC274g only decreased the deacylation

Table 1. tRNA binding by Ch-eIF2

Y-a Y-b Ss-g Kd(nM) Kd(nM) Kd(nM)

Met-tRNAfMet

(GDPNP)^a Met-tRNAfMet

(GDP)^a Met-tRNAiMet

(GDPNP)^b

1 Ch-eIF2 wt wt wt 87 ± 10 700 ± 300 27 ± 5

2 Ss-aIF2g – – wt 55 000 ± 12 000 n.d. 27 000 ± 10 000

3 Ch-eIF2bg wt wt 55 ± 20 180 ± 50 34 ± 6

4 Ch-eIF2(bN)g – bN wt 231 ± 30 557 ± 125 n.d.

5 Ch-eIF2(bC)g – bC wt 532 ± 140 1670 ± 960 n.d.

6 Ch-eIF2(bNC)g – bNC wt 3500 ± 1400 >9500 n.d.

7 Ch-eIF2ag wt – wt 9200 ± 2000 >120 000 4200 ± 2200

8 Ch-eIF2(aC274)g aC – wt 73 ± 10 740 ± 70 36 ± 8

9 Ch-eIF2(aC274)bg aC wt wt 3 ± 1 26 ± 4 n.d.

10 Ch-eIF2(aC283)g aC – wt 950 ± 180 n.d. n.d.

13 Ch-eIF2(a3)g a3 – wt >30 000 n.d. n.d.

14 Ch-eIF2(a3C274)g a3C – wt 2900 ± 500 >44 000 n.d.

Dissociation constants of Met-tRNAfMetfrom its complexes with the indicated versions of Ch-eIF2 were determined from protection experiments as described in ‘Materials and Methods’ section.

aMeasured withE. coliMet-tRNAfMetas a ligand.

bMeasured withS. cerevisiaeMet-tRNAiMet

as a ligand. n.d., not determined.

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rate of Val-tRNAfMet

UAC by 37%, consistent with a dissociation constant in the 10mM range.

In order to delineate the region responsible for the negative effect on tRNA binding, three deletions of various lengths in the C-terminal extension were performed. The three shortened proteins were named aC283, aC292 and aC298 (Figure 4). The corresponding chimeric heterodimers Ch-eIF2aCg were purified and their tRNA-binding affinities were measured. As shown in Table 1 (rows 8 and 10–12), the tRNA-binding affinity increased when the size of the acidic C-terminal extension decreased, the highest tRNA-binding affinity being obtained upon deletion of the complete acidic C-terminal extension (Table 1 and Supplementary Figure S2C). In the context of the heterotrimer, complete removal of the acidic tail of a (aC274) increased the affinity by a factor of 30 (rows 1 and 9) leading to a heterotrimer even more efficient in tRNA binding than Ch-eIF2. These results strongly suggested that the apparent weak effect of yeast eIF2a on tRNA-binding results from a positive effect of the core part of the subunit, compensated by a negative effect of the acidic residues in its appended C-terminal tail.

To conﬁrm the effect of the C-terminal tail of yeast eIF2a, we examined it using another eIF2g subunit. We therefore produced Ch-eIF2 using the central eukaryotic eIF2gsubunit fromE. cuniculi(Encc-eIF2). Two chimeric proteins were produced and puriﬁed (Figure 2, lanes 8 and 9). ChEncc-eIF2ag corresponds to the a subunit of S.

cerevisiae bound to the g subunit of E. cuniculi and ChEncc-eIF2a274Cg corresponds to the a subunit of S. cerevisiae truncated of its C-terminal tail bound to the g subunit of E. cuniculi. Dissociation constants of Met-tRNAfMet

from these proteins were determined using the same procedure as that used for yeast Ch-eIF2. Encc-eIF2g bound Met-tRNA with a Kdvalue of 1700 ± 300 nM (19). Association of yeast eIF2a to Encc-eIF2g only increased the afﬁnity of Met-tRNA by a factor of 5.6 (Table 2, rows 1 and 2) whereas the increase of afﬁnity was by a factor of 105 when eIF2aC was used in place of eIF2a(Table 2, rows 1 and 3).

Domains 1 and 2 of eIF2a contribute to tRNA-binding affinity

In the archaeal system, the aD3 domain is sufﬁcient to confer on aIF2g its full tRNA-binding afﬁnity (6,7).

Nevertheless, the 3D structure of aIF2 bound to the initiator tRNA revealed contacts of the aD12 domains with the tRNA molecule (15). To explain the apparent discrep- ancy between biochemical data and the observed contacts ofaD12 with the tRNA in the structure, it was proposed that the gain in enthalpy resulting from the contacts of the aD12 domains with the tRNA just compensates for the entropic cost of the immobilization of aD12. We anticipated that these contributions may quantitatively vary depending on theasubunit studied.

We produced the aD3 domain of yeast eIF2a. Then, tRNA-binding afﬁnity of Ch-eIF2(aD3)g heterodimer was measured. Interestingly, the tRNA-binding afﬁnity for Ch-eIF2(aD3)g heterodimer was markedly lowered

compared with that measured with the entire yeast a subunit bound to archaeal g. Only slight protection of Met-tRNA was observed at a concentration of Ch-eIF2(aD3)g heterodimer of 30mM (compare rows 7 and 13 in Table 1). This result strongly suggested that domains 1 and 2 of yeast eIF2a contribute to tRNA- binding affinity. Consistently, although deletion of the C-terminal acidic tail from aD3 increased the binding affinity of the tRNA for the chimeric heterodimer by a factor of at least 10 (compare rows 13 and 14 in Table 1), the affinity remained much lower than that measured in the presence of the aD12 domains, with the Ch-eIF2aC274gheterodimer (compare rows 14 and 8 in Table 1).

Structural studies in solution using SAXS

The region of contact between e/aIF2a and e/aIF2g mainly involves two loops of the C-terminal domain of a(aD3) as well as an elongated loop and ab-strand ofg [Figure 1A (6)]. Moreover, the structure of the human eIF2aC-terminal domain is highly similar to the structure of archaeal aIF2a (6,31,32). Therefore, besides the C-terminal extension, the structure of aD3 is conserved between eukaryotes and archaea.

In a ﬁrst step, we checked the structural similarity between the Ch-eIF2(a3C)g protein and the archaeal Ss-aIF2aD3g protein. This was achieved by measuring the X-ray scattering curve of Ch-eIF2(a3C)g. This experimental curve was then compared with the theoretical X-ray scattering curve computed from the crystallographic structure of the archaealaD3gprotein (from PDB ID 2AHO). The two curves showed a very good agreement (Supplementary Figure S3A, = 1.9). This result strongly argued in favor of a binding mode of yeast eIF2a3C protein onto Ss-aIF2g similar to that observed for the archaeal aIF2aD3 domain (6).

Finally, in order to evaluate the binding mode of the tRNA molecule by the yeast eIF2aC subunit, the scattering curve of Ch-eIF2aCg bound to Met- tRNAfMet

was measured after puriﬁcation of the complex (see ‘Materials and Methods’ section). The SAXS diffusion curve obtained with the Ch-eIF2aCg–

GDPNP–Met-tRNA_f^Met complex agreed well with the theoretical curve computed from the crystallographic structure of Ss-aIF2ag bound to Met-tRNA_f^Met (from PDB ID 3V11, Supplementary Figure S3B, = 2.8).

This strongly argued in favor of a binding mode of the tRNA by yeast eIF2a similar to the binding mode observed for the archaeal Ss-aIF2asubunit.

DISCUSSION

In this study, we have constructed Ch-eIF2 formed by assembling yeast eIF2a and b subunits to archaeal Ss-aIF2g core subunit. In such chimeric factors, the peripheral a and b subunits are likely to adopt a binding mode to the central g subunit identical to that observed for archaeal aIF2 [Figure 1A (6,10–12)]. Indeed, the binding of the bsubunit to the g one involves ana-helix of e/aIF2bwedged between twoa-helices of the G-domain

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of g (10–12). The rest of the b subunit is mobile with respect to g (10–12). Since the residues of the anchoring helix contacting thegsubunit are well conserved in all e/

aIF2b species, it is most likely that yeast eIF2bis caught by Ss-aIF2g in a similar manner. This idea is consistent with site-directed mutagenesis studies in the yeast system (33). The binding of yeast eIF2ato the archaealgsubunit was probed by SAXS (Supplementary Figure S3A). The results clearly argued in favor of a binding mode of the yeast eIF2a subunit to the archaeal g identical to that observed for the archaealasubunit.

Consistently, the chimeric protein, Ch-eIF2, is indeed able to bind yeast initiator methionyl-tRNA with a Kd

value (27 nM) similar to those measured with authentic versions of eIF2 (4,6,7,19). Moreover, we veriﬁed that

the chimeric factor remained specific for the methionine moiety of its aminoacyl-tRNA ligand. These results validated the use of such chimeric proteins to study the influence of theaandbperipheral subunits on the tRNA- binding affinity. The role of each peripheral subunit in tRNA binding was studied in the context of heterodimers (Ch-eIF2agand Ch-eIF2bg). We observe that yeast eIF2b is essential to retrieve full tRNA-binding affinity, whereas yeast eIF2a only weakly contributes to tRNA-binding affinity. These results are in keeping with the observations made using the yeast system (17,18) and shows that binding of yeast a and b subunits to archaeal g subunit confers on Ch-eIF2 a eukaryotic behavior. The same effects of the yeast a and b subunits on tRNA binding were observed when E. coli Met-tRNAfMet

was used as

Figure 4. Alignment of e/aIF2adomain 3 sequences. Representative eukaryotic (upper block) and archaeal (lower block) sequences are shown.

Aspartate and glutamate residues are boxed in gray. Secondary structures are drawn below the Ss-aIF2asequence. The positions of the last residue in C-terminal truncated versions of eIF2aare indicated.

Table 2. Effects of the C-terminal acidic tail of yeastasubunit on tRNA binding in the context ofEncephalitozoon cuniculigsubunit

Kd(nM) Kd(nM)

Y-a Encc-g Met-tRNAfMet

(GDPNP)^a Met-tRNAfMet

(GDP)^a

1 Encc-eIF2g^b – wt 1700 ± 300 >24 000

2 ChEncc-eIF2ag Wt wt 300 ± 50 >16 000

3 ChEncc-eIF2(aC)g aC wt 16 ± 1 3380 ± 550

Dissociation constants were determined from protection experiments as described in ‘Materials and Methods’ section.

aMeasured withE. coliMet-tRNAf

Metas a ligand.

bData from (19).

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the ligand instead of authentic S. cerevisiae Met- tRNAiMet

. The authentic tRNA was preferred by a factor of 2–3, whatever the combination of subunits (Table 1). This preference was also observed with the aIF2g subunit alone, consistent with the idea that the stimulatory role of the A1–U72 pair of the eukaryotic initiator tRNA is mainly exerted through binding by the g subunit (4,15,30,34). The E. coliinitiator tRNA therefore appeared as a good substitute to further dissect the structure–function relationships of the yeast eIF2a and eIF2b subunits. Hence, the bacterial tRNA was used in the rest of the study since it can be obtained with a higher purity.

The two eukaryotic-specific extensions of eIF2b posi- tively contribute to tRNA affinity. Removal of the two extensions makes a truncated eIF2b structurally homolo- gous to its archaeal counterpart. However, in contrast to archaeal aIF2b, the core-restricted version of eIF2b retains a significant ability to increase tRNA-binding affinity (by a factor of at least 28). Therefore, the eIF2b core domain contains specific features involved in tRNA- binding affinity. Overall, each of the three parts of theb subunit contributes by one order of magnitude to tRNA- binding affinity. Furthermore, these contributions are additive, thereby suggesting that the two extensions independently cooperate to enhance tRNA affinity. Whether these contributions result from direct contacts of the b subunit with tRNA or from indirect effects, for instance on the conformation of the g subunit, remains to be elucidated.

Interestingly, the near absence of effect of yeast eIF2a on tRNA affinity is only apparent. Indeed, a positive effect of the a subunit is compensated by a negative effect due to the appended eukaryotic acidic tail. The extent of the negative effect of the C-terminal tail increases with the size of the acidic peptide. TheaC274 version of yeast eIF2acontributes by at least three orders of magnitude to tRNA-binding affinity. Within this subunit, domain 3 has apparently a minor role whereas domains 1 and 2 of eIF2a bring the major contribution to tRNA affinity. Nevertheless, SAXS experiments argue in favor of a binding mode for the tRNA molecule by Ch-eIF2aCg identical to that observed within the authentic archaeal complex. The precise interactions of the domains of e/aIF2awith the tRNA molecule may explain the differences observed in the Kd measurements. In addition, at this stage, we cannot exclude that a part of the effects of theasubunit on tRNA binding may be indirect, through modulating the conformation of the g subunit, for example at the level of the switch regions.

Finally, the present study, together with those of eIF2 fromE. cuniculi(19) and archaeal aIF2 (6,7,14,35), shows that the roles of the peripheral subunits of e/aIF2 in tRNA binding vary from one organism to another. Despite this, the overall structure of the ternary initiation complex in solution is likely to remain similar in all organisms. The observed differences in the contributions of the peripheral subunits to tRNA binding might reﬂect species-speciﬁc adjustments in the mechanisms of handling of the initiator tRNA by e/aIF2 within the ribosomal initiation complex.

For example, one may propose that the negative effect of

the C-terminal tail of yeast eIF2ais cancelled on the small ribosomal subunit, thanks to an interaction with another partner of the ribosomal initiation complex. In agreement with this idea, the C-terminal tail of human eIF2a was shown to be mobile in solution (31). More generally, contacts of the a and the b subunits with other compo- nents of the initiation complex may contribute to modulate the afﬁnity of e/aIF2 for the initiator tRNA along the translation initiation process (16). Such modu- lations would, in turn, contribute to start codon recognition, in agreement with the known importance of eIF2a and eIF2bfor translation start speciﬁcity (36,37).

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online:

Supplementary Figures 1–3.

ACKNOWLEDGEMENTS

The authors thank J. Perez for expert assistance during SAXS data collection on the SWING beamline of synchrotron SOLEIL and G. Keith (Institut de Biologie Mole´culaire et Celulaire, Strasbourg) for the kind gift of yeast initiator tRNA.

FUNDING

Centre National de la Recherche Scientiﬁque; Ecole Polytechnique; Agence Nationale de la Recherche (ANR-06-PCVI-0018; MASTIC); Gaspard Monge PhD scholarship from Ecole Polytechnique (to M.N.).

Funding for open access charge: Centre National de la Recherche Scientiﬁque.

Conflict of interest statement.None declared.

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